Prothymosin alpha enhances protective immune responses induced by oral DNA vaccination against pseudorabies delivered by Salmonella choleraesuis.

Previously, we showed that vaccination with the glycoprotein D (gD) gene of pseudorabies virus (PrV) delivered by Escherichia coli induced protective immune responses. In this study, we report that oral DNA vaccination with attenuated Salmonella choleraesuis carrying the PrV gD gene conferred protective immunity in mice against PrV. Moreover, co-delivery of the prothymosin alpha gene carried by S.

Vaccination with the glycoprotein D gene of pseudorabies virus delivered by nonpathogenic Escherichia coli elicits protective immune responses.

Attenuated intracellular bacteria, such as Salmonella and Shigella, have been exploited to act as gene delivery vectors. In this study, we report that nonpathogenic, live Escherichia coli can be used for the delivery of DNA vaccines in vivo, leading to generation of immune responses against plasmid-encoded foreign antigens. The pseudorabies virus (PrV) DNA vaccine carrying the glycoprotein D (gD) gene delivered by E.

Hormone selectivity in thyroid hormone receptors.

Separate genes encode thyroid hormone receptor subtypes TRalpha (NR1A1) and TRbeta (NR1A2). Products from each of these contribute to hormone action, but the subtypes differ in tissue distribution and physiological response. Compounds that discriminate between these subtypes in vivo may be useful in treating important medical problems such as obesity and hypercholesterolemia.

Postoperative immuno-gene therapy of murine bladder tumor by in vivo administration of retroviruses expressing mouse interferon-gamma.

The murine MBT-2 bladder tumor model in syngeneic C3H/HeN mice was used to investigate the feasibility of gene therapy based on the delivery of interferon-gamma (IFN-gamma) in vivo by retroviral vectors. We constructed a recombinant retroviral vector pRUFneo/IFN-gamma, which was transfected into a retroviral packaging cell line psiCRE, to produce psiCRE/pRUFneo/IFN-gamma cells. The expressions of the neo and IFN-gamma genes were verified by reverse transcription-polymerase chain reaction and IFN-gamma was detected in the culture supernatant from psiCRE/pRUFneo/IFN-gamma cells.

Estrogen receptor pathways to AP-1.

Estrogen receptor (ER) binds to estrogen response elements in target genes and recruits a coactivator complex of CBP-pl60 that mediates stimulation of transcription. ER also activates transcription at AP-1 sites that bind the Jun/Fos transcription factors, but not ER. We review the evidence regarding mechanisms whereby ER increases the activity of Jun/Fos and propose two pathways of ER action depending on the ER (alpha or beta) and on the ligand.

Prevalence and significance of hepatitis B virus (HBV) pre-S mutants in serum and liver at different replicative stages of chronic HBV infection.

Several types of naturally occurring pre-S mutants in sera or liver tissues in patients with chronic hepatitis B virus (HBV) infection have been identified. To clarify the prevalence and significance of emergence of pre-S mutants, 140 sera and 18 resected livers from patients with HBV were studied. Replicative status was designated as high, intermediate, and low based on the HBV-DNA levels in serum or the expression of HBV antigens in liver.

Immunization with TGF-beta antisense oligonucleotide-modified autologous tumor vaccine enhances the antitumor immunity of MBT-2 tumor-bearing mice through upregulation of MHC class I and Fas expressions.

The major purpose of this study was to define if the immunosuppressive effect of a transforming growth factor-beta (TGF-beta)-producing autologous tumor vaccine can be abrogated and rendered immunogenic by suppressing its TGF-beta secretion with antisense strategy. In this study, using a TGF-beta antisense gene modified MBT-2 tumor cell line [MBT-2/TGF-beta(-)#3] which we established by ourselves, we first demonstrated that the amounts of TGF-beta produced by irradiated (IR) and non-irradiated MBT-2/TGF-beta(-) #3 were both significantly decreased when detected after in vitro culture for 48 hours. The result of flow cytometry analysis reveals that decreased production of TGF-beta led to the increased expressions of MHC class I molecule and Fas on the surface of MBT-2 tumor cells.

Postoperative administration of interleukin-12 significantly enhances the anti-tumor immune response of MBT-2 bladder cancer bearing mice.

This study, using the MBT-2 murine bladder tumor model, mainly investigated the role of interleukin-12 (IL-12) in the specific antitumor immune response of a tumor-bearing host when systemically administrated after surgery. Day 17 tumor-bearing mice (D17TBM) along with non-tumor bearing naive mice were treated with daily intraperitoneal (i.p.

The core antigen of hepatitis B virus as a carrier for immunogenic peptides.

The core antigen of hepatitis B virus (HBcAg) made in Escherichia coli yields particles that closely resemble the viral nucleocapsid. Extensive modifications can be made to the primary structure of HBcAg without impairing particle assembly. This enables other peptide sequences, including very long sequences, to be added, substituted, or inserted into the nucleocapsid subunit while retaining the ability to form highly immunogenic particles.

Hepatitis C virus core protein fused to hepatitis B virus core antigen for serological diagnosis of both hepatitis C and hepatitis B infections by ELISA.

The sequence encoding the truncated core protein (amino acids 1-98) of hepatitis C virus (HCc) was expressed in E. coli for production of HCc(1-98), or fused with the truncated core antigen (HBcAg) and segments from the preS1 and preS2 regions from hepatitis B virus (HBV) for production of HBcPreS1PreS2HCc(1-98). The HCc(1-98) and HBcPreS1PreS2HCc(1-98) proteins reacted with sera from HCV-infected individuals by immunoblot analyses, while the latter protein also exhibited HBV core antigenicity.

The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen.

Ligand-dependent activation of transcription by nuclear receptors (NRs) is mediated by interactions with coactivators. Receptor agonists promote coactivator binding, and antagonists block coactivator binding. Here we report the crystal structure of the human estrogen receptor alpha (hER alpha) ligand-binding domain (LBD) bound to both the agonist diethylstilbestrol (DES) and a peptide derived from the NR box II region of the coactivator GRIP1 and the crystal structure of the hER alpha LBD bound to the selective antagonist 4-hydroxytamoxifen (OHT).

Modulating the antitumor immunity of MBT-2 murine bladder tumor bearing mice by postoperative administration of interferon-alpha.

This study was conducted mainly to investigate the effect of interferon-alpha (IFN-alpha) on the antitumor immunity of a tumor bearing host (TBH) when postoperatively administrated with or without lethally irradiated autologous tumor cells. Using the C3H/He-MBT-2 murine bladder tumor model, a status of postoperative residual tumor was mimicked by rechallenging tumor cells 24 hours after resecting the day-17 tumor. Using immunohistochemical analysis we demonstrated that after treating with lethally irradiated MBT-2 tumor cells (IRMBT-2) + IL-2 cells of CD4+, CD8+, CD44+ and CD11b+ phenotypes prominently infiltrate the subcutaneous local injection sites.

Pro region C-terminus:protease active site interactions are critical in catalyzing the folding of alpha-lytic protease.

alpha-Lytic protease is encoded with a large (166 amino acid) N-terminal pro region that is required transiently both in vivo and in vitro for the correct folding of the protease domain [Silen, J. L. , and Agard, D.

Antisense oligonucleotide specific for transforming growth factor-beta 1 inhibit both in vitro and in vivo growth of MBT-2 murine bladder cancer.

Introduction And Objectives: TGF-beta is a potent immunosuppressive cytokine produced by many tumor cells. Secretion of TGF-beta by malignant cells may be a mechanism by which tumor cells escape destruction by tumor-specific T lymphocytes. In this study, we used a TGF-beta producing C3H/He-MBT-2 murine bladder tumor model to investigate the feasibility of antisense oligonucleotide (ODN) gene therapy strategy to block the production of TGF-beta from tumor cells and evaluate its influence on both in vitro tumor growth and in vivo tumor formation.

Beneficial effect of a bile acid resin binder on enteral feeding induced diarrhea.

Objectives: Diarrhea is a complication of enteral feeding, occurring in up to 68% of critically ill patients. We hypothesized that prolonged fasting results in abnormal bile acid homeostasis. Subsequent enteral feeding then causes a relative luminal excess of bile acids, which leads to choleretic diarrhea.

The inhibitory effect of Staphylococcus epidermidis slime on the phagocytosis of murine peritoneal macrophages is interferon-independent.

The extracellular slime produced by Staphylococcus epidermidis has been shown to interfere with several human neutrophil functions in vitro, such as chemotaxis, degranulation and phagocytosis. Slime production has been suggested as a useful marker for clinically significant infections with coagulase-negative Staphylococcus. Since the main role of macrophages in defense mechanisms is phagocytosis, the effect of slime on the phagocytic activity of macrophages was investigated.

Inhibition of alpha-lytic protease by pro region C-terminal steric occlusion of the active site.

alpha-Lytic protease, a chymotrypsin-like serine protease, is synthesized with an N-terminal 166 amino acid pro region which is absolutely required for folding of the protease. The pro region is also the most potent inhibitor of the protease known with a Ki of approximately 10(-10) M. Compared to its role in the folding reaction, relatively little is known about the mechanism by which the pro region inhibits the mature protease.

Mutated epitopes of hepatitis B surface antigen fused to the core antigen of the virus induce antibodies that react with the native surface antigen.

Fusion of peptide epitopes to the core antigen (HBcAg) of hepatitis B virus (HBV) enhances their immunogenicity, both quantitatively and qualitatively. In a number of vaccine-induced mutants of HBV, glycine145 of the surface antigen S polypeptide (HBsAg) has been replaced by arginine, resulting in loss of cross-reactivity with antibodies to normal (wild-type) HBsAg. HBcAg fusion proteins carrying the immunodominant epitope of HBsAg, in which glycine145 was replaced by arginine, glutamic acid, or lysine, were produced in Escherichia coli and formed particles that displayed HBc antigenicity and immunogenicity similar to that of HBcAg itself.

Modulation of the immunostimulating effect of autologous tumor vaccine by anti-TGF-beta antibody and interferon-alpha on murine MBT-2 bladder cancer.

Unlabelled: Our aims were to: a) elucidate whether MBT-2 cells, lethally irradiated or nonirradiated, express TGF-beta 1 mRNA and secrete TGF-beta 1 protein, and b) to investigate whether the adverse effects from IRMBT-2-secreting TGF-beta 1 in the tumor vaccine can be abrogated by exogenous addition of monoclonal anti-TGF-beta 1 antibody and/or IFN-alpha.

Materials And Methods: using the Northern hybridization analysis and the two-antibody sandwich ELISA, we demonstrate that both irradiated IRMBT-2 and nonirradiated MBT-2 cells secrete TGF-beta 1. The effect of anti-TGF-beta and/or IFN-alpha were studied by an in vitro splenocyte proliferation assay and in vivo tumor rechallenge study on day 17-TBM.

Prothymosin alpha promotes cell proliferation in NIH3T3 cells.

Thymic hormones have immunomodulatory effects on T cells and hence have been used clinically to restore the immunity of immunodeficient patients as well as to enhance the cellular immunity of cancer patients. Prothymosin alpha, which is a member of the thymic hormone family, has recently been suggested to act as a nuclear protein participating in the stimulation of cell proliferation. To characterize the biological activities ofprothymosin alpha in vitro, we established NIH3T3 cell transformants that constitutively express higher prothymosin alpha protein and its mRNA compared with the wild-type counterpart.

Determinants of performance in the isocitrate dehydrogenase of Escherichia coli.

The substrate specificity of the NADP-dependent isocitrate dehydrogenase of Escherichia coli was investigated by combining site-directed mutagenesis and utilization of alternative substrates. A comparison of the kinetics of the wild-type enzyme with 2R-malate reveals that the gamma-carboxylate of 2R,3S-isocitrate contributes a factor of 12,000,000 to enzyme performance. Analysis of kinetic data compiled for 10 enzymes and nine different substrates reveals that a factor of 1,650 can be ascribed to the hydrogen bond formed between S113 and the gamma-carboxylate of bound isocitrate, a factor of 150 to the negative charge of the gamma-carboxylate, and a factor of 50 for the gamma-methyl.

P2U purinoceptors: cDNA cloning, signal transduction mechanisms and structure-function analysis.

The cloning of a P2U purinoceptor cDNA has made it possible to use molecular biological approaches to investigate P2U purinoceptor function. Expression of recombinant P2U purinoceptors in mammalian cells lacking endogenous P2U purinoceptors has enabled us to characterize the receptor protein and its downstream effectors, and has allowed a partial analysis of the role of certain amino acid residues in ligand binding. These approaches have placed the pharmacological classification of the P2U purinoceptor on a firm molecular footing and have generated model systems that can be used to investigate receptor-ligand binding, regulation and signal transduction.

A phage display system for studying the sequence determinants of protein folding.

We have developed a phage display system that provides a means to select variants of the IgG binding domain of peptostreptococcal protein L that fold from large combinatorial libraries. The premise underlying the selection scheme is that binding of protein L to IgG requires that the protein be properly folded. Using a combination of molecular biological and biophysical methods, we show that this assumption is valid.